Expression of cell adhesion molecule in endothelial cells upon activation by human immunodeficiency virus (HIV) infection is associated with the development of atherosclerotic vasculopathy. We postulated that induction of vascular cell adhesion molecule-1 (VCAM-1) by HIV-1 Tat protein in endothelial cells might represent an early event that could culminate in inflammatory cell recruitment and vascular injury. We determined the role of HIV-1 Tat protein in VCAM-1 expression in human pulmonary artery endothelial cells (HPAEC). HIV-1 Tat protein treatment significantly increased cell-surface expression of VCAM-1 in HPAEC. Consistently, mRNA expression of VCAM-1 was also increased by HIV-1 Tat protein as measured by RT-PCR. HIV-1 Tat protein-induced VCAM-1 expression was abolished by the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) and the p38 MAPK inhibitor SB-203580. Furthermore, HIV-1 Tat protein enhanced DNA binding activity of NF-κB, facilitated nuclear translocation of NF-κB subunit p65, and increased production of reactive oxygen species (ROS). Similarly to VCAM-1 expression, HIV-1 Tat protein-induced NF-κB activation and ROS generation were abrogated by PDTC and SB-203580. These data indicate that HIV-1 Tat protein is able to induce VCAM-1 expression in HPAEC, which may represent a pivotal early molecular event in HIV-induced vascular/pulmonary injury. These data also suggest that the molecular mechanism underlying the HIV-1 Tat protein-induced VCAM-1 expression may involve ROS generation, p38 MAPK activation, and NF-κB translocation, which are the characteristics of pulmonary endothelial cell activation.

The c-Jun NH2-terminal kinases (JNK) are evolutionarily conserved serine/threonine protein kinases that are activated by proinflammatory cytokines, environmental stress, and genotoxic agents. These kinases play key regulatory roles within a cell by coordinating signals from the cell surface to nuclear transcription factors. JNK phosphorylates the amino terminal domain of all three Jun transcription factors (JunB, c-Jun and JunD) all members of the AP-1 family. The activated transcription factors modulate gene expression to generate appropriate biological responses, including cell migration, proliferation, differentiation and cell death. The role of the JNK signaling pathway in cell death/apoptosis is controversial, both pro-apoptotic and pro-survival roles have been attributed to JNK. The mechanism that enables the JNK signaling pathway to mediate both apoptosis and survival is unclear. The aim of this study is to examine the role of TNF-stimulated JNK activation on cell survival. The proinflammatory cytokine TNF, is known to activate JNK and induce apoptosis. To test whether the JNK signaling pathway contributes to TNF-induced apoptosis, the response of wild type and Jnk1-/- Jnk2-/- (JNK deficient fibroblasts) fibroblasts to TNF was examined. JNK deficient fibroblasts are more sensitive to TNF-induced apoptosis than wild-type fibroblasts. The TNF-sensitivity cannot be attributed to altered expression of TNF receptors or defects in the NF-кB or AKT pathways, known anti-apoptotic signal transduction pathways. (In fact, TNF stimulated NF-кB activation provides a major mechanism to account for survival in both wild-type and JNK deficient cells.) However this increased TNF-sensitivity can be attributed to JNK deficiency. Apoptosis is suppressed in JNK deficient cells when transduced with JNK1 retrovirus. These data implicate the JNK signaling pathway in cell survival. The AP-1 family of transcription factors is a target of the JNK signal transduction pathway. In addition JNK is required for the normal expression of the AP-1 family member, JunD. Previous studies have indicated that JunD can mediate survival. Interestingly, JNK deficient and JunD null cells display similar phenotypes: premature senescence and increased sensitivity to TNF induced apoptosis. In fact, the TNF-sensitivity is also suppressed in JNK deficient fibroblasts transduced with JunD retrovirus. Although JunD can replace the survival signaling role of JNK, phosphorylation of JunD is essential to inhibit TNF induced apoptosis. JNK deficient cells transduced with phosphomutant JunD retrovirus maintain TNF-sensitivity. Activated transcription factors modulate gene expression. It is most likely that JunD functions by regulating the expression of key molecules that act to inhibit TNF-stimulated apoptosis. Microarray analysis comparing wild-type with JNK deficient fibroblasts revealed that the expression of the survival gene, cIAP-2, was induced by TNF in only wild-type fibroblasts. Furthermore, protein expression of cIAP-2 was induced by TNF in only wild-type fibroblasts. Analysis of the cIAP-2 promoter revealed two critical NF-кB binding sites and one AP-1 binding site. Luciferase reporter assays indicated key roles for both NF-кB and the AP-1 component, JunD in TNF-induced cIAP-2 gene expression. These experiments establish that the JNK/JunD pathway collaborates with NF-кB pathway to increase the expression of the anti-apoptotic protein cIAP-2 in TNF treated cells. Without this collaboration, the JNK pathway mediates apoptosis. The integration of JNK signaling with other signaling pathways represents a mechanism to account for the dual ability of the JNK pathway to mediate either survival or apoptosis. The dynamic coordination of signals within and between pathways is critical. The future challenge will be to fit the details of individual signaling pathways into the context of signaling networks.

Tyrosine phosphorylation of proteins by protein tyrosine kinases is an important step in initiating mitogenic signal transduction pathways. The receptor tyrosine kinases represent a class of protein kinases that employ phosphorylation cascades to transmit a signal generated at the cell surface. The AP-1 transcription factor is a common target of receptor tyrosine kinase activation, transformation by Ras-like proteins and activation of the MAP kinase pathway. The AP-1 complex contains a dimer of Jun proteins or a heterodimer of Jun and Fos or other bZip proteins. The transcriptional activation of Jun is enhanced by phosphorylation on residues Ser-63 and Ser-73. Therefore, identifying the regulatory proteins kinases of Jun would be an important link in signaling from the upstream cell surface events to downstream events, such as gene expression. The JNK1 protein kinase was identified and phosphorylates c-Jun at these sites. The JNK1 protein is a member of the JNK group of protein kinases, which are activated in response to UV treatment. JNK1 is the 46 kDa isoform, and the isolation of the 55 kDa isoform is described in this thesis. Furthermore, a role for JNK was established in Drosophila. Drosphila JNK (DJNK) is essential for the process of dorsal closure. The JNK protein kinases are involved in cytokine signaling, response to environmental stress and development.

Stress-induced JNK activity has been implicated in apoptosis. Gene disruption studies have established that JNK signaling is required for some forms of apoptosis. However, it was not clear whether and how JNK was able to deliver an apoptotic signal, because JNK and its regulated-downstream transcriptional factors control a variety of gene activities and multiple biological functions. I have studied this question by using constitutively activated JNK that is independent of upstream signaling. The results indicate that activated JNK is sufficient to deliver an apoptotic signal that causes cytochrome c release from mitochondria. Significantly, this apoptotic signal requires pro-apoptotic Bc12 proteins of Bax and Bak to mediate the downstream apoptotic program. This part of work established the apoptotic activity of JNK signal transduction and the key downstream components of JNK-stimulated apoptotic signal. Two pathways are known to mediate apoptosis in response to apoptotic stimulations: death receptor pathway and mitochondrial pathway. It has been established that JNK is required for the apoptosis mediated by mitochondria in response to ultraviolet irradiation and some genetic stress. However, the mechanisms are not fully understood. It is well known that Bax and Bak are indispensable downstream components leading to apoptotic mitochondrial changes and that other Bc12 family members can regulate the relative apoptotic activity of Bax and Bak. In conjunction with the first part of the research, I have investigated the hypothesis that JNK-mediated regulation of BH3-only Bc12 members contributes to its apoptotic activity. These results indicate that JNK-mediated phosphorylation of Bim and Bmf promotes the release of these proapoptotic BH3-only proteins from their sequestration and these factors become free to initiate apoptosis. This part of work established one mechanism of activated JNK-stimulated apoptosis. This mechanism may contribute to the phenomenon that Jnk1-/-Jnk2-/- fibroblasts are resistant to ultraviolet irradiation-induced apoptosis.

cJun NH2-terminal kinase (JNK) is a member of the MAPK (mitogen- activated protein kinase) signaling family that responds to various extracellular stimuli, such as stress, growth factors, cytokines, or UV radiation. JNK activation can lead to cellular responses including gene expression, growth, survival, and apoptosis. JNK has been implicated in normal developmental processes, including tissue morphogenesis, as well as pathological processes, such as cellular transformation and cancer. JNK exists in three isoforms, and knockout mice have been generated for each isoform; the ubiquitously expressed Jnk1 and Jnk2 have been studied independently, however, the two isoforms are partially functionally redundant. Jnk1-/- Jnk2-/-mice are nonviable, therefore studies of compound JNK-deficiency have been limited to mouse embryonic fibroblasts (MEF). Understanding the role of JNK in epithelial cells is now possible with the creation of conditional JNK knockout animals. I sought to elucidate the role of JNK in cellular transformation, cancer, and normal development. I employed both in vitro and in vivo approaches. First, I evaluated the role of JNK in cellular transformation using p53-/- Jnk1-/- Jnk2-/- MEF transduced with oncogenic Ras. To extend this study, I examined JNK-deficiency in a Kras-induced model of lung tumorigenesis. Second, I investigated JNK1- and JNK2-deficiency in a p53-mediated model of mammary tumorigenesis. Finally, I examined the role of JNK in mouse mammary gland development by establishing JNK-deficient primary mouse mammary epithelial cells and evaluating JNK-deficient mammary gland transplants. Taken together, this work provides evidence of context-dependent roles for JNK in both normal and pathological cell biology.

The central nervous system (CNS) is composed of two types of cells: neurons that send electrical signals to transmit information throughout the animal and glial cells. Glial cells were long thought to be merely support cells for the neurons; however, recent work has identified many critical roles for these cells during development and in the mature animal. In the CNS, glial cells act as the resident immune cell and they are responsible for the clearance of dead or dying material. After neuronal injury or death, glial cells become reactive, exhibiting dramatic changes in morphology and patterns of gene expression and ultimately engulfing neuronal debris. This rapid clearance of degenerating neuronal material is thought to be crucial for suppression of inflammation and promotion of functional recovery, but molecular pathways mediating these engulfment events remain poorly defined. Drosophila melanogaster is a genetically tractable model system in which to study glial biology. It has been shown that Drosophila glia rapidly respond to axonal injury both morphologically and molecularly and that they ultimately phagocytose the degenerating axonal debris. This glial response to axonal debris requires the engulfment receptor Draper and downstream signaling molecules dCed-6, Shark, and Rac1. However, much remains unknown about the molecular details of this response. In this thesis I show that Drosophila c-Jun kinase (dJNK) signaling is a critical in vivo mediator of glial engulfment activity. In response to axotomy, glial dJNK signals through a cascade involving the upstream MAPKKKs Slipper and TAK1, the MAPKK MKK4, and ultimately the Drosophila AP-1 transcriptional complex composed of JRA and Kayak to initiate glial phagocytosis of degenerating axons. Interestingly, loss of dJNK also blocked injury-induced up-regulation of Draper levels in glia and glial-specific over-expression of Draper was sufficient to rescue phenotypes associated with loss of dJNK signaling. I have identified the dJNK pathway as a novel mediator of glial engulfment activity and show that a primary role for the glial Slipper/Tak1→MKK4→dJNK→dAP-1 signaling cascade is activation of draper expression after axon injury.

No description available.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12969号 / 論医博第2102号 / 新制||医||1012(附属図書館) / 32407 / 新制||医||1012 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山田 泰広, 教授 福田 和彦, 教授 伊達 洋至 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

The current studies were designed to explore the protective effects of pifithrin-α and melatonin against doxorubicin-induced cardiotoxicity. Doxorubicin was injected at a dose of 22.5 mg/kg (i.p.) in mice to induce cardiotoxic effects. Meanwhile, doxorubicin caused a significant increase of cardiac cell apoptosis following injection (14.2 ± 1.1% for doxorubicin-5 d vs. 1.8 ± 0.12% for control, P < 0.01). Ribonuclease protection assays and Western blot analyses revealed that doxorubicin upregulated the p53-dependent genes Bax, BclxL, and MDM2 at least 2-fold. p53 was phosphorylated at Ser 15 in mouse hearts 1 h following doxorubicin injection, and p38 and ERK1/2 MAPKs mediated the phosphorylation of p53. In addition, caspases-3 and -9 were activated 24 h after doxorubicin injection. A p53 inhibitor, pifithrin-α, inhibited doxorubicin-induced apoptosis when administered at a dose of 2.2 mg/kg. Pifithrin-α abolished p53 transactivation activity, but did not influence doxorubicin-induced phosphorylation at Ser 15. By effectively inhibiting the expression of p53-dependent genes, pifithrin-α blocked doxorubicin-induced activation of caspases-3 and -9, thereby preventing cardiac apoptosis. In addition, pifithrin-α attenuated doxorubicin-induced structural and functional damages, without diminishing its anti-tumor efficacy on p53-null PC-3 cancer cells. The protective effects of melatonin and its metabolite 6-hydroxymelatonin on doxorubicin-induced cardiac dysfunction were evaluated in an isolated perfused mouse hearts and in vivo doxorubicin-treated mice. While perfusion of mouse hearts with 5 μM doxorubicin for 60 min resulted in a 50% suppression of HRxLVDP and a 50% reduction of coronary flow, pre-exposure of hearts to 1 μM melatonin or 6-hydroxymelatonin eased the cardiac dysfunction. In addition, administration of melatonin or 6-hydroxymelatonin (2 mg/kg/d) significantly attenuated doxorubicin-induced cardiac dysfunction, myocardial lesions, and cardiac cell apoptosis. Melatonin and 6-hydroxymelatonin significantly improved the survival rate of doxorubicin-treated mice. Another melatonin analog, 8-methoxy-2-propionamidotetralin, did not show any convincing protection on either animal survival or on in vitro cardiac function, presumably due to its lack of free radical-scavenging activity. Finally, neither melatonin nor 6-hydroxymelatonin compromised the anti-tumor activity of doxorubicin in cultured PC-3 cells. These studies suggest that pifithrin-α and melatonin have significant therapeutic potential for patients suffering doxorubicin-induced cardiotoxicity.